The elucidation of molecular alterations that occur during human breast cancer development may permit the identification of preventative strategies for women at high risk. Lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) confer 4- and 8-10 fold increases in risk for the development of invasive carcinoma, respectively. Our goal is to identify proteins whose expression levels vary between normal ductal/lobular units and premalignant/DCIS lesions using laser capture microdissection and proteomics. Using flash frozen double mastectomy material containing DCIS, we have processed 2D gels of DCIS, ipsilateral normal and contralateral normal breast. Eighteen differentially expressed protein spots have been collected for mass spec sequencing. Other similar samples are currently being processed. Each protein identified will be tested in a larger cohort of premalignant/DCIS and infiltrating ductal breast lesions. We examined a cohort of approximately 100 human biopsy specimens using in situ hybridization to examine trends in mRNA expression levels, with the goal of identifying genes whose expression patterns correlated with increasing risk. Two genes, cyclin D and the RXR form of the retinoic acid receptor superfamily, exhibited quantitatively increased mRNA levels in DCIS specimens than in normal ductal/lobular units in the margin of the specimen. Overexpression of these two genes therefore correlates with increased risk for development of invasive breast carcinoma. Cyclin D has been transfected into the human immortal MCF-10A breast cell line. Increased anchorage-independent growth was observed, without full tumorigenicity, suggesting that cyclin D1 overexpression can contribute, but is insufficient for full tumorigenicity. Interestingly, the anchorage-independent growth of cyclin D transfectants was several fold to a log more senstive to the inhibitory effects of g-irradiation than the control transfectants. This inhibition was accompanied by increased apoptosis, and could be replicated using the apoptosis inducer Apo-2 (TRAIL), but not using TNF-a. The cyclin D1 transfectants exhibited altered levels of functional Apo-2 receptors following radiation, which may contribute to their apoptotic response. Antibody to Apo-2 partially inhibited the radiation effect on colonization. We conclude that cyclin D overexpression can produce colonization-competent cells in the breast, and that certain apoptosis inducers may hold promise for eliminating such cells. If confirmed and extended, this may constitute a new approach to prevention.